The PuhB Protein of Rhodobacter capsulatus Functions in Photosynthetic Reaction Center Assembly with a Secondary Effect on Light-Harvesting Complex 1

Abstract

The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.Purple nonsulfur photosynthetic bacteria such as Rhodobacter capsulatus are capable of aerobic respiratory and anaerobic photosynthetic growth. The photosynthetic apparatus includes three membrane-bound pigment-protein complexes: the reaction center (RC), which functions as a light-dependent quinone reductase; light-harvesting complex 1 (LH1), which is adjacent to and forms a ring or arc encircling the RC as part of the so-called core complex that includes the PufX polypeptide (17,22,36,38,43,51); and LH2, which is thought to be present in multiple copies of a ring-shaped structure that interconnect core complexes (33). These complexes are located within differentiated invaginations of the cytoplasmic membrane, called the intracytoplasmic membrane system, formed in response to oxygen deprivation (14). The presence of the various photosynthetic complexes can be evaluated by their characteristic light absorption spectra, which reflect the protein environments around bacteriochlorophyll a (BChl); unbound BChl has a far-red absorption peak at 780 nm, whereas this peak of the LH2 BChls of R. capsulatus is at 800 and 850 nm, and BChls in the LH1 complex absorb light at approximately 870 nm. The far-red absorption peaks of the RC are at about 760 nm (bacteriopheophytins), 804 nm (accessory or "voyeur" BChls), and 865 nm (the "special pair" of BChls) (16).In R. capsulatus, one of the three polypeptides of the RC, RC H, is encoded by the puhA gene that is transcribed as part of the bchFNBHLM-lhaA-puhABC superoperon from two promoters, one 5Ј of bchF and the other within the lhaA gene (3,6,57). A segment of the puh operon is shown in Fig. 1a. The PuhB protein (formerly known as Orf214) is required for optimal RC/LH1 l...

Transcriptome Dynamics during the Transition from Anaerobic Photosynthesis to Aerobic Respiration in Rhodobacter sphaeroides 2.4.1

“…The rapidity with which fbcQ transcription falls off is to be contrasted with the more moderate decreases observed for fbcB, suggesting that the Q subunit is not essential for the aerobic functioning of the bc 1 complex. The result is consistent with previous studies showing that fbcQ is not expressed under highly aerated conditions and a After R. sphaeroides 2.4.1 was grown anaerobically by sparging it with 95% N 2 -5% CO 2 in the light (10 W/m 2 ) to an OD 600 of 0.086, the growth conditions were shifted to aerobic-dark by sparging with 69% N 2 -30% O 2 -1% CO 2 .…”

Investigation of Rhodobacter capsulatus PufX interactions in the core complex of the photosynthetic apparatus

“…E. coli MM39, a kind gift of J. Beckwith, was used for the TOXCAT system (Russ and Engelman 1999). Plasmids pTB999, pTPR9, pTPR8, pMA10, pStu I, pcc-KAN, pccTNM, pccGpAwt, and pccGpA83I have been described previously (Klug and Cohen 1988;Zilsel et al 1989;Richter and Drews 1991;Russ and Engelman 1999;Aklujkar et al 2005a). These bacterial strains and plasmids, and others constructed in this study, are listed in Table 1.…”

“…The wild-type R. capsulatus strain SB1003 (Solioz and Marrs 1977), its LH2 ) derivative MW442 (Scolnik and Marrs 1987), and RC ) LH1 ) LH2 ) derivatives of MW442, U43 (Youvan et al 1985) and MA01 (Aklujkar et al 2005a), which are PufX ) and PufX + , respectively, have been described previously. MA15 is an RC ) LH1 ) -LH2 + PufX + strain derived from SB1003 (Aklujkar et al 2005b).…”

“…MA15 is an RC ) LH1 ) -LH2 + PufX + strain derived from SB1003 (Aklujkar et al 2005b). Escherichia coli strain HB101 (pRK2013) (Ditta et al 1985) was used to transfer plasmids conjugatively from E. coli C600 (Bibb and Cohen 1982) to R. capsulatus as previously described (Aklujkar et al 2005a). E. coli MM39, a kind gift of J. Beckwith, was used for the TOXCAT system (Russ and Engelman 1999).…”

“…Measurements of the BChl peaks characteristic of the RC special pair (865 nm) and LH1 (880 nm) were carried out as described (Aklujkar et al 2005a). Similarly, the area of the RC voyeur BChl peak (804 nm) was computed from 800 to 830 nm to minimize the contribution of BPhe and unbound BChl, and the baseline subtracted to correct for light-scattering was drawn from 700 to 830 nm.…”